Current quantitative methods and assays, such as immunoassays, for determining the presence and amount of specific analytes in a sample are limited to the recognition of a single antigen (analyte) per solid phase. Generally, such quantitative methods are limited to the use of a solid phase, such as a plate containing microwells or a membrane that have only one specific antibody immobilized thereto. See US 2016/0289308; US 2016/0297893. Typically, the antibodies used in such assays are labeled (e.g., fluorescent, radioactive, luminescent, secondary antibody) in a manner that renders them detectable (or not) once they bind an antigen in the sample. Hence, in order to quantitatively detect more than one analyte in a sample, assays currently employ the use of separate solid phase components each having a single antibody specific for one particular analyte immobilized thereto.
Previous attempts to utilize a single solid phase to detect multiple analytes have been directed to the use of lateral flow assays (see, e.g., US 2016/0289308; US 2016/0297893; and US 2010/061377). Lateral flow assays require a test sample to flow in a chromatographic fashion along a bibulous or non-bibulous porous solid phase, such a membrane. In a typical, lateral flow assay a sample is applied to the solid phase at a first location (i.e., an application zone) and transits the solid phase until the sample reaches a second discrete location (a first test zone) on the solid phase, which includes a first antibody specific to a first analyte. The first test zone is then analyzed to determine the presence or amount of a first analyte in the sample. The sample then must flow until it reaches a third discrete location (second test zone) on the solid phase, which includes another antibody that is specific to another analyte. In a lateral flow assay, the second test zone is then analyzed to determine the presence or amount of analyte in the sample. Determination of the presence of analytes in a lateral flow assay requires a detectable signal at each discrete location (test zone) on the solid phase, which is then read by an instrument such as a fluorometer. However, in order to decipher the amount of each analyte detected in each zone distinct labels are typically used, such as different wavelengths of light, different fluorescent dye labels or different labeled secondary antibodies. As such, lateral flow detection assays are expensive, technically complex and prone to operator based errors.
Solid phase analyte immunoassays are often able to “detect” the presence of multiple analytes in a sample but only in a qualitative fashion. In some instances, solid phase analyte immunoassays include a solid phase having multiple analytes immobilized thereto can be quantitative, but these are limited to competitive assays. For example, in solid phase analyte immunoassays, analytes not antibodies are bound to a solid phase. The bound analytes compete with analyte present in a sample for binding to a labeled antibody specific for the analyte of interest. In these assays, the sample (and antibody bound thereto) is removed in a wash step resulting in a loss of signal compared to that of a solid phase that containing immobilized analytes that are all bound to a detectable antibody (i.e., a control). Hence, the presence of an analyte in a sample is deduced from a reduction signal when compared to a control. As stated above, detection of multiple analytes using a single solid phase requires differentially labeled antibodies and requires the application and reapplication of a sample and specific antibodies to the solid phase. The application of multiple labeled antibodies and subsequent wash steps that are required by such applications make the competitive solid phase assays inefficient, labor intensive and costly. Additionally, competitive assays are susceptible to the non-complexed labeled antibodies binding to “immunoreactive” polypeptide species present in the sample instead of the analyte of interest, and thus prone to false positives.
Solid phase immunoassays for the detection of multiple analytes through the use of a solid phase with multiple antibodies attached thereto are generally qualitative. For example, when a sample is provided to a solid phase that incorporates a plurality of antibodies, each of which is specific to a different antigen of interest is contacted with a sample; if the sample contains any of the antigens of interest a signal would be produced. However, the assay is incapable of deciphering between which of the numerous antigens of interest are present in the sample, without different distinct labels and means for detecting each distinct label. Further, the signal obtained in such assays is a composite of the signals produced by all the different bound antigens. Hence, current methods are incapable of quantitatively determining how much of a specific antigen of interest is present in a sample without using a separate solid phase for each analyte of interest.
In view of the foregoing, the requirements for producing separate solid phase components specific to each analyte assayed renders multi-analyte assays imprecise, expensive, technically complex, and time-consuming. Therefore, a need exists for methods that enable the quantitative detection of multiple analytes in a sample using a single solid phase.